C-terminal peptide α-thioesters are key intermediates in the synthesis of small and medium-sized proteins and cyclic peptides by native chemical ligation. These mildly activated species are also required for the construction of topologically and backbone engineered proteins.
C-terminal peptide α-thioesters can be prepared by standard solid-phase peptide synthesis (SPPS) using Boc/benzyl chemistry, or for larger polypeptide domains and protein domains, using intein-based bacterial expression systems. The Boc/benzyl approach requires the use of anhydrous HF which is not well suited for synthesis of phospho- and glyco-peptides. In addition, anhydrous HF is very toxic and requires special equipment for handling.
The Fmoc-based methodology is attractive as it does not employ HF and hence provides the synthesis of phospho- and glyco-peptides in good yields. However, the poor stability of the thioester functionality to strong nucleophiles such as piperidine, which is used for the deprotection of the Nα-Fmoc group, seriously limits the use of this methodology for the preparation of peptide α-thioesters. So far, several approaches have been used to overcome this limitation. Futaki et al. used an approach where peptide α-thioesters were prepared in solution using a partially protected precursor. (See Futaki, S.; Sogawa, K.; Maruyama, J.; Asahara, T.; Niwa, M. Tetrahedron Lett. 1997, 38, 6237.) Li et al. used a Fmoc-deprotection cocktail compatible with α-thioesters to synthesize an unprotected 25-residue peptide α-thioester in moderate yield. (See Li, X. Q.; Kawakmi, T.; Aimoto, S. Tetrahedron Lett. 1998, 39, 8669.) A similar approach was also used by Clippingdale et al. using in this case a non-nucleophilic base in combination with 1-hydroxybenzotriazole (HOBt). (See Clippingdale, A. B.; Barrow, C. J.; Wade, J. D. J. Pept. Sci. 2000, 6, 225.)
Alternatively, the introduction of the α-thioester function at the end of a synthesis has been used by Alsina et al. where the backbone amide linker (BAL) was employed for the synthesis of peptide thioesters using an Fmoc-based strategy. This approach was used for the synthesis of small peptide thioesters in good yields. However, some racemization was observed during the thiolysis step. Swinnen et al used the phenylacetamidomethyl (PAM) and Wang resins to synthesize peptide α-thioesters by employing EtSH in the presence of Me2AlCl to effect thiolysis of the resin-bound peptide. This approach was used for the synthesis of a 22-residue peptide α-thioester in moderate yield. Another approach developed by Ingenito et al. and Shin et al. involved the use of Kenner's sulfonamide safety-catch linker. This linker is fully stable to repetitive exposure to the basic conditions needed for Fmoc deprotection. When the sulfonamide is alkylated, the peptide resin is activated and easily cleaved with thiols to yield the corresponding peptide α-thioester. However, the use of akylating agents (such as CH2N2 or ICH2CN) has been shown to alkylate unprotected methionine residues. More recently, Brask et al. have introduced a new method for the generation of peptide thioesters using a trithioortho ester linker. (See Brask, J.; Albericio, F.; Jensen, K. J. Org. Lett. 2003, 5, 2951.)